Kazumi Akimoto

HMG-CoA Reductase Inhibitor Increases GTP Cyclohydrolase I mRNA and Tetrahydrobiopterin in Vascular Endothelial Cells

Objective—Endothelial nitric oxide synthase (eNOS) activity is supported by tetrahydrobiopterin (BH4), which appears to be important for generating protective NO but decreases uncoupling formation of superoxide. We investigated the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, in terms of BH4 metabolism in human umbilical vein endothelial cells (HUVECs). Methods and Results—We measured the mRNA levels of GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the first step of de novo BH4 synthesis, by real-time polymerase chain reaction. The mRNA of GTPCH, as well as of eNOS, was upregulated in HUVECs treated with cerivastatin. This increase was time and dose dependent. Fluvastatin was also observed to enhance GTPCH and eNOS mRNA levels. In parallel with this observation, cerivastatin increased intracellular BH4. Incubating HUVECs with tumor necrosis factor (TNF-&agr;) was observed to increase GTPCH mRNA while decreasing eNOS mRNA. In the presence of cerivastatin, the TNF-&agr;–mediated increase in GTPCH mRNA was enhanced, and the TNF-&agr;–mediated decrease in eNOS mRNA was attenuated. Cerivastatin increased the stability of eNOS mRNA. However, it did not alter the stability of GTPCH mRNA but increased GTPCH gene transcription, as shown by nuclear run-on assays. Preteatment of HUVECs with the selective GTPCH inhibitor, 2,4-diamino-6-hydroxypyrimidine, caused a decrease in intracellular BH4 and decreased citrulline formation after stimulation with ionomycin. Furthermore, the potentiating effect of cerivastatin was decreased by limiting the cellular availability of BH4. Conclusions—Our data demonstrate that statins elevate GTPCH mRNA, thereby increasing BH4 levels in vascular endothelial cells. In addition to augmenting eNOS expression, statins potentiate GTPCH gene expression and BH4 synthesis, thereby increasing NO production and preventing relative shortages of BH4.

RETRACTED ARTICLE: Angiotensin-II-induced oxidative stress elicits hypoadiponectinaemia in rats

Aims/hypothesisHypertension, endothelial dysfunction and insulin resistance are associated conditions that share oxidative stress and vascular inflammation as common features. Adiponectin is an abundant plasma adipokine that plays a physiological role in modulating lipid metabolism and exerts a potent anti-inflammatory activity. We hypothesised that adiponectin levels decrease in response to oxidative stress and that this may promote the development of hypertension, endothelial dysfunction and insulin resistance.MethodsRats were infused with angiotensin II (AngII) or its vehicle, either alone or in combination with tempo1 (4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl), a membrane-permeable metal-independent superoxide dismutase mimetic, or tetrahydrobiopterin (BH4), one of the most potent naturally occurring reducing agents and an essential cofactor for nitric oxide synthase activity. Heart rate, systolic blood pressure, body weight and serum levels of adiponectin were measured on day 7 of treatment, and then the animals were killed. Vessel tone and superoxide production were measured ex vivo in thoracic vascular rings. The expression of adiponectin mRNA in adipose tissue was assessed by Northern blotting, and in 3T3-L1 adipocytes exposed to H2O2 by real-time PCR. The expression of NAD(P)H oxidase subunit mRNAs in the rats was assessed by RT-PCR and real-time PCR.ResultsHypertension and endothelial dysfunction were induced in rats by infusion of AngII and reversed by administration of tempol. Plasma concentrations of adiponectin and adipose tissue levels of adiponectin mRNA were decreased in AngII-infused rats, and this effect was prevented by cotreatment with tempol or BH4. The production of superoxide anions (O2−) was significantly increased in the aortae of AngII-treated rats, and this increase was prevented by the administration of tempol or BH4. Levels of mRNAs that encode NAD(P)H oxidase components, including p22phox, gp91phox, p47phox and Rac1, were similarly increased in adipose tissue, aortae and hearts of AngII-infused rats. Cotreatment of rats with tempol or BH4 reversed AngII-induced increases in NAD(P)H oxidase subunit mRNAs. Fully differentiated 3T3-L1 adipocytes, also exhibited diminished adiponectin mRNA levels when exposed to low concentrations of H2O2.Conclusions/interpretationOur results demonstrate that AngII-induced oxidative stress and endothelial dysfunction are accompanied by a decrease in adiponectin gene expression. Since antioxidants were observed to prevent the actions of AngII, and H2O2 on its own suppressed adiponectin expression, we conclude that adiponectin gene expression is negatively modulated by oxidative stress. Plasma adiponectin levels may provide a useful indicator of oxidative stress in vivo, and suppressed levels may contribute to the proinflammatory and metabolic derangements associated with type 2 diabetes, coronary artery disease and the metabolic syndrome.

Activation of macrophages by lactoferrin: secretion of TNF‐α, IL‐8 and NO

When macrophages were cultured with lactoferrin, cytokines such as tumor necrosis factor (TNF‐α), interleukin 8 (IL‐8) and nitric oxide (NO) were secreted. Secretion of TNF‐α peaked at 6 h of incubation in the presence of lactoferrin and then declined. About 80% of the maximum secretion of IL‐8 was observed at 6 h of incubation. The concentration of IL‐8 in the culture medium remained almost constant between 24‐72 h. In contrast, no siginificant effect on NO secretion was observed at 6 h, but a significant effect was observed at 24 h and secretion gradually increased between 24‐72 h. The effects of lactoferrin on the secretion of TNF‐α, IL‐8 and NO were dose‐dependent and lactoferrin had a significant effect on secretion at concentrations greater than 10 mg/ml. The use of reverse transcription‐polymerase chain reaction (RT‐PCR) showed that the results obtained were consistant with the cytokine secretion results. It is concluded that lactoferrin activates macrophages which result in the secretion of TNF‐α, IL‐8 and NO.

Fasudil, a Rho-kinase inhibitor, attenuates glomerulosclerosis in Dahl salt-sensitive rats.

Objective The present study was designed to clarify whether the Rho–Rho-kinase pathway is involved in the process of hypertensive glomerulosclerosis and to assess the therapeutic effect of fasudil, a specific Rho-kinase inhibitor. Method and results Dahl salt-sensitive rats (DS) and Dahl salt-resistant rats (DR) were fed a high-salt diet at 6 weeks of age. Fasudil (30 mg/kg per day) was administered for 7 weeks to DS starting at the age of 11 weeks. After 7 weeks, untreated DS were characterized by decreased kidney function, increased proteinuria, abnormal morphological findings, increased adrenomedullin and atrial natriuretic peptide (ANP) levels, and increased renal messenger RNA expression of RhoB, Rho-kinaseα, Rho-kinaseβ, collagen I and collagen III, and transforming growth factor-beta (TGF-β) in the renal cortex compared with DR. Chronic fasudil treatment significantly improved renal function (serum creatinine, –26%; blood urea nitrogen, –41%; creatinine clearance, +42%), proteinuria (–24%) and histological findings (glomerular injury score, –49%; afferent arteriolar injury score, –17%) without changing blood pressure compared with untreated DS. Interestingly, long-term fasudil treatment decreased the plasma adrenomedullin (–25%) and ANP (–49%), but did not change the plasma renin or aldosterone. Furthermore, fasudil significantly decreased the messenger RNA expression of TGF-β (–20%), collagen I (–23%), and collagen III (–24%) in the renal cortex. However, there were still significant differences in the aforementioned parameters between DR and fasudil-treated DS. Conclusion These results suggest that the Rho–Rho-kinase pathway may be partly responsible for the pathogenesis of hypertensive glomerulosclerosis independently of blood pressure in DS, and that chronic inhibition of the Rho–Rho-kinase pathway may be a new strategy for treating hypertensive nephrosclerosis.

Renoprotective Effect of Chronic Adrenomedullin Infusion in Dahl Salt-Sensitive Rats

The present study was designed to examine whether chronic adrenomedullin infusion has renoprotective effects in hypertensive renal failure and the mechanism by which chronic adrenomedullin infusion exerts its effects. Dahl salt-sensitive rats and Dahl salt-resistant rats were fed a high salt diet starting at 6 weeks of age. Recombinant human adrenomedullin or vehicle was infused for 7 weeks in 11-week-old Dahl salt-sensitive rats. Dahl salt-resistant rat was used as a control. After 7 weeks, untreated Dahl salt-sensitive rats were characterized by decreased kidney function, abnormal morphological findings, increased hormone levels, increased renal tissue angiotensin II levels, and altered mRNA expressions of transforming growth factor &bgr; (TGF-&bgr;) and components of the renin-angiotensin system compared with Dahl salt-resistant rats. Chronic adrenomedullin treatment significantly improved renal function (serum creatinine −87%, creatinine clearance +114%, urinary protein excretion −59%) and histological findings (glomerular injury score −54%) without changing mean arterial pressure compared with untreated Dahl salt-sensitive rats. Interestingly, long-term human adrenomedullin infusion decreased the endogenous rat adrenomedullin level (−97%) with a slight increase of human adrenomedullin level. Chronic adrenomedullin treatment also significantly inhibited the increase of plasma renin concentration (−269%), aldosterone level (−82%), and renal tissue angiotensin II levels (−60%). Furthermore, adrenomedullin infusion significantly decreased the increases of mRNA expressions of TGF-&bgr; (− 63%), angiotensin-converting enzyme (−137%), renin (−230%), and angiotensinogen (−38%) in renal cortex. These results suggest that increased endogenous adrenomedullin plays a compensatory role in chronic hypertensive renal failure and that long-term adrenomedullin infusion has renoprotective effects in this type of hypertension model, partly via inhibition of the circulating and renal renin-angiotensin system.

Globular Adiponectin Activates Nuclear Factor-κB and Activating Protein-1 and Enhances Angiotensin II–Induced Proliferation in Cardiac Fibroblasts

Adiponectin is present in the serum as a trimer, hexamer, or high–molecular weight form. A proteolytic cleavage product of adiponectin, known as globular adiponectin (gAd), also circulates in human plasma. The biological activities of these isoforms are not well characterized. Pressure overload in adiponectin-deficient mice results in enhanced concentric cardiac hypertrophy and increased mortality, suggesting that adiponectin inhibits hypertrophic signaling in the myocardium. Therefore, we examined whether gAd exerts the same effects on myocardium signaling. Nuclear factor-κB (NF-κB) and activating protein-1 (AP-1) activation were examined using cardiac fibroblasts prepared from the ventricles of 1- to 2-day-old Wistar rats and grown in culture. gAd activated NF-κB and enhanced tumor necrosis factor-α (TNF-α)-induced NF-κB activity. gAd also activated AP-1 and enhanced angiotensin II (Ang II)-induced AP-1 activity. gAd induced mRNA expression of c-fos and c-jun and activated extracellular signal–regulated kinase. Thus, gAd enhanced Ang II–induced DNA and collagen synthesis. Antibodies against adiponectin receptor (AdipoR)1 and AdipoR2 elicit activation of NF-κB or AP-1, two redox-sensitive transcription factors. Thus, rather than having an antihypertrophic effect, gAd might contribute to the activation of myocardium signaling, leading to myocardial hypertrophy.

Long-Term Administration of Rho-Kinase Inhibitor Ameliorates Renal Damage in Malignant Hypertensive Rats

We have shown recently that fasudil, a Rho-kinase inhibitor, has renoprotective effects in salt-sensitive hypertensive rats. We hypothesized that activation of Rho-kinase is involved in the pathogenesis of glomerulosclerosis in malignant hypertensive rats. To test this hypothesis, we studied the following 4 groups: control Wistar–Kyoto rats, untreated deoxycorticosterone-acetate salt spontaneously hypertensive rats (DOCA-SHR), low-dose fasudil-treated DOCA-SHR, and high-dose fasudil-treated DOCA-SHR. After 3 weeks of treatment, the effects of fasudil were examined. DOCA-SHR was characterized by increased blood pressure (BP); increased kidney weight; decreased renal function; increased proteinuria; abnormal histological findings; increased monocyte/macrophage infiltration; increased urinary 8-isoprostran levels; increased gene expression of collagen I, collagen III, transforming growth factor-β, and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits (p40phox, p47phox, and p67phox); and decreased gene expression of endothelial NO synthase (eNOS) in the renal cortex as compared with Wistar–Kyoto rats. Long-term high-dose fasudil treatment significantly improved renal function and histological findings without changing BP, as compared with untreated DOCA-SHR. Interestingly, long-term fasudil treatment significantly decreased monocyte/macrophage infiltration and urinary 8-isoprostran excretion, in association with decreased mRNA levels of transforming growth factor-β, collagen I, collagen III, and NADPH oxidase subunits (p40phox, p47phox, and p67phox), and increased mRNA levels of eNOS in the renal cortex. Long-term low-dose fasudil treatment tended to improve these variables slightly but did not affect most of them significantly. Our results suggest that long-term fasudil treatment provides renoprotective effects independent of BP-lowering activity. These renoprotective effects are associated with inhibition of extracellular matrix gene expression, monocyte/macrophage infiltration, oxidative stress, and upregulation of eNOS gene expression.

Cilostazol inhibits cytokine-induced nuclear factor-κB activation via AMP-activated protein kinase activation in vascular endothelial cells

AIMS Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cyclic AMP (cAMP) levels and activates protein kinase A, thereby inhibiting platelet aggregation and inducing peripheral vasodilation. We hypothesized that cilostazol may prevent inflammatory cytokine induced-nuclear factor (NF)-kappaB activation by activating AMP-activated protein kinase (AMPK) in vascular endothelial cells. METHODS AND RESULTS Cilostazol was observed to activate AMPK and its downstream target, acetyl-CoA carboxylase, in human umbilical vein endothelial cells (HUVEC). Phosphorylation of AMPK with cilostazol was not affected by co-treatment with an adenylate cyclase inhibitor, SQ 22536, and a cell-permeable cAMP analogue, pCTP-cAMP, did not induce AMPK phosphorylation and had no effect on cilostazol-induced AMPK phosphorylation, suggesting that cilostazol-induced AMPK activation occurs through a signalling pathway independent of cyclic AMP. Cilostazol also dose-dependently inhibited tumour necrosis factor alpha (TNFalpha)-induced NF-kappaB activation and TNFalpha-induced I kappa B kinase activity. Furthermore, cilostazol attenuated the TNFalpha-induced gene expression of various pro-inflammatory and cell adhesion molecules, such as vascular cell adhesion molecule-1, E-selectin, intercellular adhesion molecule-1, monocyte chemoattractant protein-1 (MCP-1), and PECAM-1 in HUVEC. RNA interference of AMPK alpha 1 or the AMPK inhibitor compound C attenuated cilostazol-induced inhibition of NF-kappaB activation by TNFalpha. CONCLUSION In the light of these findings, we suggest that cilostazol might attenuate the cytokine-induced expression of adhesion molecule genes by inhibiting NF-kappaB following AMPK activation.

Chronic administration of adrenomedullin attenuates transition from left ventricular hypertrophy to heart failure in rats

Abstract—Acute administration of adrenomedullin (AM) exerts beneficial hemodynamic, renal, and neurohormonal effects in heart failure (HF). However, chronic effects of AM administration on HF remain unknown. This study sought to examine the effect of chronic infusion of AM on progression of HF in rat. Human recombinant AM was administered by osmotic minipump for 7 weeks in the HF model of Dahl salt-sensitive rats. The effect was compared with vehicle and diuretic treatment group. Chronic AM infusion significantly decreased left ventricular end-diastolic pressure, right ventricular systolic pressure, right atrial pressure, and left ventricular weight/body weight (P <0.01 for all). AM significantly attenuated the increase in circulating renin-aldosterone, endogenous rat AM, and atrial natriuretic peptide levels (P <0.01 for all). AM also inhibited the myocardial tissue levels of angiotensin II and atrial and brain natriuretic peptide (P <0.01 for all). These changes were associated with the improvement of cardiac output and systemic vascular resistance (both P <0.05). Furthermore, AM improved left ventricular end-systolic elastance (P <0.01). These improvements were greater in the AM than in the diuretic group, although both drugs similarly decreased systolic blood pressure and increased urinary sodium excretion. Kaplan-Meier survival analysis showed that AM significantly prolonged survival time compared with diuretic (P <0.05) and vehicle (P <0.01) treatment groups. These results suggest that endogenous AM plays a compensatory role in HF and that chronic AM infusion attenuates progression of left ventricular dysfunction and improves survival, at least in part, through inhibition of circulating and myocardial neurohormonal activation.

Glucose upregulates plasminogen activator inhibitor–1 gene expression in vascular smooth muscle cells

We investigated the effects of high concentrations of glucose on plasminogen activator inhibitor-1 (PAI-1) gene expression in cultured rat vascular smooth muscle cells (VSMC). In response to a high glucose concentration (27.5 mM), PAI-1 mRNA increased within 2 h, peaked at 4 h, remained elevated for another 4 h, then decreased to basal levels at 24 h. On the other hand, mannose at the same concentration (22.5 mM mannose plus 5.5 mM glucose) as an osmotic control had little effect on PAI-1 mRNA expression. The expression of PAI-1 mRNA that was also increased by H(2)O(2), angiotensin II, or phorbol myristate acetate, was reversed by the MAPK kinase (MEK) inhibitor PD98059 or the specific protein kinase C (PKC) inhibitor GF109203X. High glucose appeared to activate MAPK and PKC in VSMC judging from Elk-1 and AP-1 activation, respectively. PD98059 inhibited and GF109203X prevented subsequent PAI-1 induction by glucose. These results suggest that glucose at high concentrations induces PAI-1 gene expression in VSMC at least partially via MAPK and PKC activation. This direct effect of glucose might have important implications for the increased plasma concentrations of PAI-1 and possibly atherosclerosis that are associated with diabetes.